Browsing by Author "Gillespie, Patrick"

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    Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism
    (Elsevier, 2023) Lancaster, Makayla S.; Kim, Byungwook; Doud, Emma H.; Tate, Mason D.; Sharify, Ahmad D.; Gao, Hongyu; Chen, Duojiao; Simpson, Ed; Gillespie, Patrick; Chu, Xiaona; Miller, Marcus J.; Wang, Yue; Liu, Yunlong; Mosley, Amber L.; Kim, Jungsu; Graham, Brett H.; Medical and Molecular Genetics, School of Medicine
    Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.
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    Sucla2 Knock‐Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type–Specific Phenotypes
    (Wiley, 2024) Lancaster, Makayla S.; Hafen, Paul; Law, Andrew S.; Matias, Catalina; Meyer, Timothy; Fischer, Kathryn; Miller, Marcus; Hao, Chunhai; Gillespie, Patrick; McKinzie, David; Brault, Jeffrey J.; Graham, Brett H.; Medical and Molecular Genetics, School of Medicine
    Background: Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel in vivo model of mitochondrial myopathy. Methods: The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model in vivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, ex vivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Results: Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. Conclusions: SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in ex vivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.
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    Untargeted Metabolomics of Slc13a5 Deficiency Reveal Critical Liver–Brain Axis for Lipid Homeostasis
    (MDPI, 2022-04-14) Milosavljevic, Sofia; Glinton, Kevin E.; Li, Xiqi; Medeiros, Cláudia; Gillespie, Patrick; Seavitt, John R.; Graham, Brett H.; Elsea, Sarah H.; Medical and Molecular Genetics, School of Medicine
    Though biallelic variants in SLC13A5 are known to cause severe encephalopathy, the mechanism of this disease is poorly understood. SLC13A5 protein deficiency reduces citrate transport into the cell. Downstream abnormalities in fatty acid synthesis and energy generation have been described, though biochemical signs of these perturbations are inconsistent across SLC13A5 deficiency patients. To investigate SLC13A5-related disorders, we performed untargeted metabolic analyses on the liver, brain, and serum from a Slc13a5-deficient mouse model. Metabolomic data were analyzed using the connect-the-dots (CTD) methodology and were compared to plasma and CSF metabolomics from SLC13A5-deficient patients. Mice homozygous for the Slc13a5tm1b/tm1b null allele had perturbations in fatty acids, bile acids, and energy metabolites in all tissues examined. Further analyses demonstrated that for several of these molecules, the ratio of their relative tissue concentrations differed widely in the knockout mouse, suggesting that deficiency of Slc13a5 impacts the biosynthesis and flux of metabolites between tissues. Similar findings were observed in patient biofluids, indicating altered transport and/or flux of molecules involved in energy, fatty acid, nucleotide, and bile acid metabolism. Deficiency of SLC13A5 likely causes a broader state of metabolic dysregulation than previously recognized, particularly regarding lipid synthesis, storage, and metabolism, supporting SLC13A5 deficiency as a lipid disorder.